1). Immunology of CML stem cells: Non-cycling, quiescent CD34+ CML cells are more resistant to tyrosine kinase inhibitors. We have addressed the question of whether this small population of true leukemia stem cells (also characterized by the CD34+CD38-Lin-CD90+ phenotype), which represent a reservoir of relapsing leukemia, are susceptible to immune attack by T cells and NK cells. In summary we identified a number of leukemia antigens that could be targets for T cell attack: minor histocompatibility antigens (mHAgs) HA1 and SMCY and leukemia associated antigens(LAA) WT1, PR3, NE, PRAME, BMI-1. We examined the effect of NK cells on cycling and quiescent CD34+ CML cells. We found that the proteasome inhibitor Bortezomib significantly enhanced autologous NK cells cytotoxicity on cycling and quiescent non-cycling CD34+ CML cells by 20-40%. These findings support the use of LAA vaccines or LAA specific T cells or alternatively the development of allogeneic NK cell therapy for CML. Currently there are no active protocols to explore these approaches. 2) NK cells in myeloid malignancies: (Dr Kate Stringaris) In a multivariate analysis, studying established factors predictive of relapse after SCT, and exploring known associations of KIR-mismatching with recipient HLA class I molecules, we found that donors with KIR types KIR 2DS1, 3DS1 and 2 DL5A gave a significantly greater probability of remaining in remission in AML recipients. Further laboratory studies exploring KIR polymorphisms in AML are ongoing in collaboration with Dr Kate Stringaris who has continued these studies at the Hammersmith Hospital London. 3) Immune profiling in AML: To further explore NK and T cell function in AML we have established A collaboration with Dr Stephen Strickland, (Vanderbilt University Hospital, Nashville TN) the immune profile of patients with AML are studied at presentation, after remission induction, and at relapse. We are exploring the hypothesis that at presentation immune function is compromised by the leukemia through one or more mechanisms that suppress antileukemia responses. We are monitor immune recovery and the circulating cytokines that drive lymphocyte recovery, characterizing the NK and T cell phenotypes in remission, studying NK function (cytotoxicity against standard targets and autologous blasts) and T cell function (frequency and cytotoxic characteristics of T cells recognizing LSA peptide mixtures including WT1, neutrophil elastase, Aurora kinase, MAGE 3 and PRAME). We have analyzed 20 patients. Preliminary findings indicate an increase in effector T reg cells at presentation and remission and the occurrence of AML blasts with myeloid-derived suppressor cell function suggesting that immune responses to AML may be blunted by a negative immune milieu. Findings will be correlated with patient outcome to identify favorable immune recovery patterns that protect against relapse. In a separate study we have analyzed the potential of patients with AML in remission to generate LAA specific T cells. In comparison with their HLA identical stem cell transplant donors we found that patients possess a comparable but different repertoire of cytotoxic T cells recognizing a panel of LAA. These findings support the development of LAA-specific T cell in the autologous setting to maintain remission in AML patients. Ultimately these studies should identify protective immune mechanisms in AML that inform rational immunotherapy (vaccination, blocking suppressor cells, cytokine treatment, NK or T cell adoptive therapy).&#8232; 4) MDS: We have shown in prior reports that patients with MDS have a variety of immune abnormalities associated with a T cell mediated suppression of marrow function. We recently completed a trial of Campath immunosuppression to restore hematological function in early , low grade MDS. We showed a 77% response rate in 22 evaluable MDS intermediate-1 patients and a 57% response rate in 7 evaluable intermediate-2 patients at 3 months. We have now addressed the problem of MDS patients with more advanced disease in transformation to AML. These patients have a poor prognosis. We have initiated a trial exploring the association of low dose clofarabine as chemotherapeutic agent with lenalidomide immune stimulation. This trial recently opened to accrual. 5) Large granular lymphoproliferative disease (LGL): Previous studies showed that some patients with LGL respond to CSA. To explore whether more intensive immunosuppression would be more effective at restoring hematopoiesis, patients with LGL were given alemtuzemab (anti CD52 monoclonal). Fifteen of 19 patients responded rapidly. All patients had a sustained and prolonged lymphopenia but without developing opportunistic infection. There was a massive reduction of the LGL clones in responders when evaluated at 3 and 6 months. Relapse was associated with resurgence of the clone. We have confirmed that about 40-50% of LGL patients have mutations in stat-3 . However there appears to be no prognostic relationship between stat-3 mutation or CD52 expression on the LGL cell and treatment outcome. We are now using the stat-3 mutation to probe LGL T cell subsets to identify the earliest clonal progenitor of the LGL. Selected LGL samples whose CD8 T cells are over 95% clonal are being screened against a combinatorial peptide library to identify the cognate peptides and attempt to identify the target protein (collaboration with Dr M Wooldridge, Cardiff, UK). Understanding how the LGL T cell suppresses myelopoiesis and erythropoiesis may shed light on the mechanism of suppression of both normal and leukemic cells by cytotoxic T cell populations.